We propose to study how cytochrome c oxidase, which has subunits encoded in both the mitochondrion and nucleus, is assembled in the inner membrane. This process is likely to be important to human disease because it has become clear recently that both aging and many OXPHOS diseases exhibit cytochrome c oxidase deficiencies. These are degenerative tissue specific diseases that include: fatal and benign infantile myopathies, Leigh's syndrome, ischemic heart disease, Alzheimer's disease, and Parkinson's disease. The precise cause of these diseases is unknown. Although many OXPHOS diseases have been linked to mitochondrial genes it has become clear recently that their genetics is complex and that both nuclear and mitochondrial genes are involved. To begin to address this problem we will first study the assembly of yeast cytochrome c oxidase. The biogenesis of this protein requires 3 mitochondrial genes and at least 38 nuclear genes. Several recent studies have suggested that the assembly of this multimeric protein is facilitated by a number of proteins, including -both heat shock proteins and cytochrome c oxidase specific """"""""assembly facilitator"""""""" proteins. Our overall objectives are to elucidate the sequence in which subunits assemble,~ identify partial complexes that are intermediates in the process, and determine how cytochrome c oxidase specific """"""""assembly facilitator"""""""" proteins function in this pathway. Our experimental strategy will combine the use of a collection of novel """"""""assembly defective"""""""" pet mutants, conditional mutants in these genes, conditional and null mutants in COX structural genes, in vivo kinetic analyses, biochemical and immunochemical fractionation studies, a synchronized in vivo assembly system, and a newly developed mitochondrial in vitro system that supports import, export, and assembly. Once we have established a role for """"""""assembly facilitator"""""""" proteins in yeast we will ask if similar proteins function in the assembly of human cytochrome c oxidase. This will be approached by cloning human genes that functionally complement well-understood """"""""assembly defective"""""""" yeast mutants. The availability of these human genes should provide an opportunity to begin to address the molecular basis for cytochrome c oxidase defects in OXPHOS diseases.
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